Phosphocholine linked prodrug derivatives

ABSTRACT

Disclosed are compounds of general formula (I) that function as prodrugs, thereby increasing bioavailabilities of the linked therapeutic agents, wherein the LINKER is (i) substituted or unsubstituted alkyl, (ii) substituted or unsubstituted alkenyl, (iii) substituted or unsubstituted alkanoyl, (iv) substituted or unsubstituted alkenoyl wherein the double bond is cis, and (v) (ortho or para) carbonyl-substituted aryl; and wherein the subtituent is each an independent group or linked together thereby forming a ring; and wherein X is one or more substituted or unsubstituted group containing one or more O, N, or S atom and wherein the substituent is each an independent group or linked together thereby forming a ring; and wherein the therapeutic agent is an alcohol-containing water-insoluble steroids or another alcohol containing compounds and methods to prepare such compounds.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a 371 of International Application No.PCT/US00/04140, filed Feb. 16, 2000, which claims priority of U.S.Provisional Patent Application No. 60/120,483, filed Feb. 18, 1999, theentire disclosure of which is incorporated by reference herein.

FIELD OF THE INVENTION

This invention pertains to methods and compositions for increasing theaqueous solubility and bio-availability of bioactive agents byconjugating them to phospholipids.

BACKGROUND OF THE INVENTION

Conventional means for delivering pharmaceutical and therapeutic agentsto mammals often are severely limited by chemical and physical barriersto uptake, as well as by susceptibility of administered agents to rapidmetabolic inactivation following uptake. Oral delivery of manybiologically-active agents would be the route of choice if not for theextreme pH of the stomach, the action of proteolytic and other digestiveenzymes in the intestine, and the impermeability of gastrointestinalmembranes to the active ingredient.

Methods for orally administering vulnerable pharmacological agents haverelied on co-administration of adjuvants (e.g. resorcinols and non-ionicsurfactants such as polyoxyethylene oleyl ether and n-hexadecylpolyethylene ether) to artificially increase the permeability of theintestinal walls; co-administration of enzymatic inhibitors (e.g.pancreatic trypsin inhibitor, diisopropylfluorophosphate (DFP) andtrasylol) to avoid enzymatic degradation; and encapsulation of theactive agent in liposomes or other delivery vehicles.

Irrespective of the mode of administration of many therapeuticcompounds, once they gain access to body tissues or fluids they are thensubject to rapid inactivation in the liver, termed the first-passeffect. Orally administered compounds in particular are rapidlydelivered to the liver via the portal circulation. Many compounds areacted upon by mixed-function oxidases, Phase I enzymes and other liverenzymes to produce inactive glucuronides, hippurates, glycyl and acetylderivatives, which are rapidly excreted by the kidney.

There is thus a need in the art for methods and compositions to enablepotential therapeutic agents to be rapidly absorbed in the intestine andavoid first-pass inactivation in the liver.

SUMMARY OF THE INVENTION

It has now been unexpectedly discovered that conjugation of manybiologically active agents to phospholipid via a phosphodiester bondwill significantly enhance the bioactivity and/or the bioavailability ofsuch agents.

In one aspect, the present invention provides a method for increasingthe bioavailability of a pharmaceutical agent, comprising the steps ofconjugating said agent to one or more phospholipid moieties, recoveringsaid biologically active agent conjugated to said phosphocholine andadministering said agent to a mammal wherein said agent in conjugatedform is significantly more soluble in aqueous media than said agent inunconjugated form.

In yet another aspect, the present invention provides a composition ofmatter comprising an isolated phospholipid derivative of salicylic acid.

In yet another aspect, the present invention provides a pharmaceuticalformulation for treating a mammal suffering from osteoporosis comprisingan isolated phospholipid derivative of a compound selected from thegroup consisting of estrone or estradiol and a pharmaceuticallyacceptable carrier or diluents.

In yet another aspect, the present invention provides a composition ofmatter comprising an isolated phospholipid derivative of an antibioticselected from the group consisting of cephalosporin P1, fusidic acid andhelvolic acid.

In yet another aspect, the present invention provides a composition ofmatter comprising an isolated phospholipid derivative ofdehydroepiandosterone.

These and other aspects of the present invention will be apparent tothose of ordinary skill in the art in light of the present description,claims and drawings.

DETAILED DESCRIPTION OF THE INVENTION

All patent applications, patents, and literature references cited inthis specification are hereby incorporated by reference in theirentirety. In case of inconsistencies, the present description, includingdefinitions, will prevail.

Definitions

“Phospholipid-conjugated” or “phospholipid-derivatized” defined hereinas covalently bonded to a phospholipid moiety via a phosphodiesterlinkage.

“Significantly enhanced bioactivity” or “significantly more soluble inaqueous media” in terms of the conjugated drugs of the present inventionis defined herein as no less than 5 to 10-fold increased biologicalactivity and/or aqueous solubility as compared to the unconjugatedparent compound when administered by the same route.

The present invention is directed to increasing the bioavailabilityand/or aqueous solubility of pharmaceutically active agents,specifically by conjugation of such agents to phospholipids, such as aphosphocholine moiety via a phosphodiester bond.

In accordance with the present invention, therapeutic substances willbenefit by increasing their water solubility (and their bioavailability)by forming a phosphodiester between an (a) alcohol, and (b) aphospholipid. Non-limiting examples of the phospholipid includephosphocholine, phosphoserine, phosphotyrosine, phosphoethanolamine,n-monoakyl-phosphoethanolamine and N, N-dialkyl-phosphoethanolamine (allcommercially available from Aldrich Chemical, Milwaukee, Wis.).Phosphocholine is particularly preferred as the phospholipid.

Phosphocholine is a ubiquitous component of biological membranes,usually present in the form of phosphatidyl choline, i.e., attached viaa phosphodiester bond to diacyl glycerol. The two most commonphosphocholine-containing molecules are lecithin and sphingomyelin. Bothof these compounds can be hydrolyzed by phospholipase C at thephosphocholine phosphodiester bond to release diacyl glycerol andceramides, respectively. Importantly, both lecithin and sphingomyelin,which are present in food, are absorbed in the gastrointestinal tract,incorporated into HDL- and LDL-cholesterol, and transported through theblood without significant first-pass metabolism in the liver.

In accordance with the present invention, conjugation of one or morephospholipid moieties to lipophilic compounds will render them morehydrophilic, without abrogating their ability to traverse biologicalmembranes. Without wishing to be bound by theory, it is contemplatedthat phospholipid conjugation will, in most cases, mask the biologicalactivity of the conjugated compounds. The phospholipid conjugates willpersist in conjugated form until they encounter enzymes such asphospholipase C, sphingomyelinase and non-specific esterases, which aremembers of the signal transduction pathway (Methods in Enzymotofy, Vol.197, E. Dennis, editor, Academic Press, NY) and are present in thecirculation and on target tissues. These enzymes will then remove thephospholipid moiety and liberate the original compound with itsbiological activity intact. The above-mentioned enzymes are specific forphosphocholine; other esterases of the signal transduction system wouldhydrolyze the other phosphoesters (Methods in Enzymology, Vol. 201, T.Hunter, Academic Press, NY, Beth Sefton, editor). In this manner,addition of phospholipid is expected to protect compounds fromfirst-pass inactivation in the liver and allow them to reach their sitesof action in the blood or in peripheral tissues.

Pharmaceutical agents suitable for use in the present invention include,without limitation, lipophilic compounds that exhibit poor solubility inbiological fluids, as well as compounds that are rapidly metabolized inthe liver to hippurate, glucuronate, or other derivatives. Non-limitingexamples of suitable compounds include those that are not presentlyutilized in pharmaceutical applications, in particular as orallyadministrable agents, because of problems with solubility, uptake,and/or metabolism. The only requirements for an agent to be used in thepresent invention are 1) the presence of a free alcohol functional groupto which a phospholipid may be attached, and 2) the susceptibility ofthe resulting phosphodiester bond to cleavage by phospholipase C,sphingomyelinase or other mammalian esterases.

Examples of pharmaceutical agents suitable for use in the presentinvention include without limitation steroids, catecholamines such asepinephrine or norepinephrine, prostaglandins such as prostaglandin E1or E2, leukotrienes such as leukotriene B4, C4 or D4 and peptides.Peptides for use in the present invention are those which contain serineor threonine and preferably should not be longer than 10–15 amino acidresidues in length such as Leutinizing Hormone Releasing Hormone (LHRH)(a 10 amino acid peptide) and its analogues. Preferred startingcompounds or pharmacological agents include testosterone (available fromSigma, St. Louis, Mo.), etiocholanolone (Sigma), estradiol (Sigma),estrone (Sigma) and dehydroepiandrosterone (Sigma). These steroids haveonly limited activity when administered orally.

In an alternative embodiment of the present invention antibiotics, suchas cephalosporin P1, can be conjugated to phospholipids in order toincrease its aqueous solubility and decrease it metabolism on the firstpass through the liver and excretion on the first pass through thekidney. Non-limiting examples of compounds for use in this embodiment ofthe present invention include cephalosporin P1 (isolated as described inBurton et al., Biochem. J. 50:168–174, 1951; Halsall et al., Chem.Comm., pp. 685–687, 1966), fusidic acid (commercially available fromSigma), and helvolic acid (commercially available from Sigma). Use ofthese antibiotics has been limited because of an inability todevelopment therapeutic serum and tissue levels in recipient mammalsand, perhaps, because of the ease of development of resistance. Theapparent resistance may be caused by induction of metabolic enzymes asoccurs with other steroidal therapeutic agents.

Non-limiting examples of additional substances for use in the presentinvention containing a free alcohol group include the steroidalsubstances mentioned above (DHEA, eticholanolone, testosterone,estradiol, estrone, catecholamines, etc.), the antibiotics mentionedabove, aglycones including cardiac glycosides, such as digoxigenin(commercially available from Sigma), digitoxigenin (commerciallyavailable from Sigma), ouabagenin (commercially available from Sigma)and salicylic acid (commercially available from Sigma).

Presented below is a further list of non-limiting examples of compoundsfor use in the present invention. Following the name of the compound,presented in parentheses is the number assigned to the compound in theMerck Index, 1996, 12th Edition. Menadiol (5873), Metronidazole (6242),Clindamycin (2414), Pentaerythritol Tetranitrate (7249), Mesalamine(5964), β-Tocopherol (9632), γ-Tocopherol (9633), δ-Tocopherol (9634),Roxindole (8432), Vitamin E (10159), Styramate (9027), Strophanthidin(9015), Vitamin A (10150), Vitamin D₂ (10156), Vitamin D₃ (10157),Vitamin A₂ (10151), Calcitriol (1681), Diflunisal (3190), ClavulanicAcid (2402), Retinoic Acid (8333), Mazindole (5801).

A compound particularly well-suited for use in the present invention isthe cyclic Urea-based HIV-1 protease inhibitor DMP-323 (J. Med. Chem.39:2156–2169, 1996). Due to its low aqueous solubility investigatorsfound that there was variability in the compounds bioavailability uponadministration to patients and inconsistent efficacy. Addition of aphospholipid moiety is expected to improve its therapeutic use.

Other compounds well-suited for use in the present invention includeaglycones from cardiac glycosides such as digoxigenin, digitoxigenin andouabagenin (all commercially available from Sigma, St. Louis, Mo.).

In addition to increasing the solubility of the above-identifiedcompounds, the primary effect of conjugation to a phospholipid moiety tothe following water soluble compounds is expected to be an increasedhalf-life, that is to say, they will be long-acting forms of the parentcompounds. Non-limiting examples of such compounds include Isoproterenol(5236), Propranolol (8025), Methyldopa (6132), Epinephrine (3656),Codine (2525), Codine Phosphate (2528), Acetaminophen (45), Aspirin(886).

The conjugated therapeutic agents will be at least ten times more watersoluble then the original alcohol. This will increase theirbioavailability and decrease their metabolism to, e.g., the 3-glycosidein the case of steroids, which should be a major excretion pathway. Thedecreased glycoside formation will be caused by the presence of thephosphoester at that site. The derivative is not expected to be activeprior to hydrolysis of the phospholipid group. The present inventor hasfound that lymphocytes have an enzyme on their cell membrane thatcleaves phosphocholine from other compounds (for example, sphingomyelinor lecithin) to release phosphocholine and the other ester conjugate(ceramide or diacylglycerol). The activity of this enzyme is stimulatedten-fold by TGF-α (data not shown). Without wishing to be bound bytheory, it is believed that use of phospholipid-conjugated antibioticsof the present invention will lead to high concentrations of activeagents at the site of an infection by the following mechanism.Lymphocytes are attracted to the site of an infection or inflammationwhere they release TGF-α, which, in turn, stimulates phospholipidhydrolysis in other subtypes. This same process will lead to localrelease of an active form of the antibiotic from the phospholipiddiester conjugate. Because of the response of the enzyme to localconcentrations of TGF-α, there should be a correspondingly high localconcentration of the antibiotic. This will lead to effective therapy andlower toxicity.

According to the present invention, starting compounds may be convertedto phospholipid derivatives using any methods that are known in the art.In one preferred embodiment, phosphocholine (obtainable from SigmaChemicals, St. Louis, Mo.) is reacted with a soluble carbodiimide,preferably 1-ethyl-3(3-dimethyl-aminopropyl)carbodiimide hydrochloride(EDAC, Sigma) in an active ester condensation reaction. Thiscarbodiimide is preferred because it, similar to phosphocholine, iswater-soluble. The active phosphoester intermediate is then reacted witha pharmaceutically active agent to yield the desired phosphocholineester. The reaction is shown in Example 1 below. Phosphocholine in wateris reacted with EDAC to yield the active ester. This is then reactedwith, e.g., testosterone or other biologically active starting compoundsetc., to yield the final product

or other active esterification product. The product is expected to beessentially water-soluble and thus easily separated from the startingcompound by conventional extraction and/or separation methods e.g. FlashChromatography, Thin Layer Chromatography, High Performance LiquidChromatography (HPLC) and the like, as is known to those of ordinaryskill in the art.

Alternate methods for synthesis of phosphocholine derivatives includephosphorylation of the steroid, peptide, etc. with DPPP to give aphosphate ester, e.g., testosterone phosphate, which is coupled tocholine using EDAC as the complexing agent.

Alternately, the alcohol (“drug”) may be reacted with phosphorousoxychloride and the aminoalcohol component added in excess. In this wayall of the unreacted phosphorous oxychloride will be used up. Thephosphochloride ester intermediate can also be isolated and reacted as asecond step with the amino-alcohol component (choline, etc.). The finalproducts can be purified by HPLC.

The phospholipid derivatized drugs of the present invention are expectedto demonstrate enhanced biological activities, increased bioavailabilityand increased aqueous solubility. For example, etiocholanolone ismetabolized by formation of the glucuronide in the liver of a mammal.After oral administration, about 99% of all free etiocholanolone isinactivated on each pass through the liver. When etiocholanolone isorally administered, it is absorbed in the gastrointestinal tract andtransported via the portal circulation directly to the liver.Subsequently, only a fraction of a percent of the administered drug isbiologically available for function. In contrast,phosphocholine-conjugated etiocholanolone may bind to form Low DensityLipoprotein (LDL) and High Density Lipoprotein (HDL) cholesterol and isnot expected to be degraded on first passage through the liver. In itsphosphocholine-derivatized form, it is believed that about 80% of theetiocholanolone would not be metabolized at each pass. When thephosphocholine moiety is removed by an esterase, such as phospholipaseC, sphingomyelinase, etc., then the parent compound will be availablefor binding and function in the target tissue. Glucuronidation wouldonly occur on its return to the liver after removal of thephosphocholine moiety.

The phospholipid-conjugated compounds of the present invention may beadministered therapeutically by any route known in the art, e.g.,orally, intravenously, intramuscularly, subcutaneously, by inhalation orin aerosol form, and topically. The present invention is particularlyapplicable to compounds that, in their unconjugated state, cannot beeffectively administered by the oral route.

The phospholipid-conjugated compounds of the present invention can betested for efficacy as follows. A starting compound, and itsphospholipid derivative, may be administered by any of the above routesto a test animal, e.g., rat, mouse, rabbit, guinea pig, and the like.Serum samples are then collected at increasing times afteradministration, and the levels of the starting and conjugated compoundare assayed and compared. It will be understood by those skilled in theart that the method of assay will depend upon the starting compound. Inthe case of steroids or peptides, High-Performance LiquidChromatography, Thin-Layer Chromatography, or immunoassay may be used toquantify serum levels. When the starting compounds are gonadal steroids,it may also be necessary to gonadectomize the test animals prior to drugadministration, so as to suppress endogenous production of the testcompound. Successful compounds are those whose serum level is increasedsignificantly by administration of the phospholipid derivative relativeto administration of the starting compound or by their ability to reachtherapeutically significant serum levels when administered by analternate route, e.g. orally.

In a second phase, the starting compound and its phospholipid derivativewill be administered to test animals, and the physiological effect ofthe compounds assayed over time. For example, for etiocholanolone andits phospholipid derivative(s), rate of weight gain and changes in basalmetabolic rate are measured. Estradiol, estrone and their phosphocholinederivatives will be administered by gavage to ovariectomized mice orrats and changes in uterine weight, breast development and estradiolblood levels will be measured. Testosterone and its phosphocholinederivative will be administered orally to castrate mice or rats andchanges in seminal vesicles, prostate size, and levator and muscle willbe determined. Theophylline and its phosphocholine derivatives will begiven orally to rats and the blood levels over the next 6 hours will bedetermined. From these tests, the degree to which the phospholipidderivatives are more potent than the underivatized parent compound willbe determined, i.e., the same response will be achieved with a smallerdose of the derivatized compound than the parent compound. This will bea measure of greater potency. Successful compounds are those whosefunctional endpoints are significantly lower for phospholipidderivatives than for the starting compounds.

In a preferred embodiment of the present invention, testosterone isconverted to testosterone-17-phosphocholine, estrone is converted toestrone-3-phosphocholine and estradiol is converted toestradiol-3-phosphocholine or estradiol-17-phosphocholine. In likemanner, theophylline is converted to theophylline phosphocholine. Thesecompounds will frequently be given as replacement therapy for varioushormone deficiencies and as pharmacological therapies in other cases.Theophylline is given to treat asthma, estradiol is administered totreat osteoporosis, etiocholanolone is given as a haemapoetic agent, topromote weight loss and to reduce diabetic blood sugar levels. Similarderivatives could also be used to provide enhanced levels ofepinephrine.

The present invention also provides pharmaceutical formulations anddosage forms comprising the phospholipid-derivatized drugs of thepresent invention. The pharmaceutical formulations of the presentinvention may also include, as optional ingredients, pharmaceuticallyacceptable vehicles, carriers, diluents, solubilizing or emulsifyingagents, and salts of the type well known to those of ordinary skill inthe art.

The phospholipid-derivatized drugs of the present invention can beincorporated into pharmaceutical formulations to be used to treatmammals. Pharmaceutical formulations comprising thephospholipid-conjugated drugs of the present invention as at least oneof the active ingredients, would in addition optionally comprisepharmaceutically-acceptable carriers, diluents, fillers, salts and othermaterials well-known in the art depending upon the dosage form utilized.For example, preferred parenteral dosage forms may comprise a sterileisotonic saline solution, 0.5 N sodium chloride, 5% dextrose and thelike. Methyl cellulose or carboxymethyl cellulose may be employed inoral dosage forms as suspending agents in buffered saline or incyclodextran solutions to enhance solubility.

It will be appreciated that the unit content of active ingredient oringredients contained in an individual dose or dosage form need not initself constitute an effective amount for the various usages of thephospholipid-derivatized drugs of the present invention since thenecessary effective amount can be reached by administration of aplurality of such dosage forms.

The following examples are intended to further illustrate the presentinvention without limiting it thereof.

EXAMPLE 1 Synthesis of Phosphocholine Derivatives Method 1

Phosphocholine (Sigma) (0.1 mol) is stirred in pyridine (Fisher, VWR)(100 ml) with 0.1 mol of morpholine (Sigma) and 0.1 mol of DDC (Sigma)for 6 hours under nitrogen or argon. At this point the reaction complexis stirred while 0.1 mol of steroid (etiocholanolone, estradiol,testosterone) are added. After stirring for an additional 3 hours thereaction mixture is diluted with 1 liter of ice water. The insolubleN,N′ dicyclohexylurea is removed by filtration and the aqueous fractionis extracted with 4×0.5 volumes of ethyl acetate. The ethyl acetate iswashed with saturated brine (0.1 vol) to remove the pyridine and driedover sodium sulfate. The solvent is removed by filtration and theproduct isolated by LH-20 column chromatography or by preparative HPLC.

Method 2

Phosphocholine (0.1 mol), steroid (0.1 mol) as above and DCC (0.12 mol)are stirred in 100 ml of pyridine (VWR) at 80° for 6 hours undernitrogen. The solution is diluted with 600 ml of water and processed asdescribed above.

Method 3

Testosterone or other steroid, prostaglandin, etc. (0.1 mol) is reactedwith POCl₃ in pyridine to yield the steroid phosphate. This productafter drying in pyridine will then be reacted with 0.1 mol of EDAC at arate just sufficient to maintain the pH at 7.0. The product is thenpurified as described above.

The compounds will then be analyzed by HPLC to determine purity of thereaction product, by NMR to verify the structure and by UV and IRspectra to determine their identity. Treatment with a phosphodiesterasewill then be used to cleave the diester to further establish thestructural identity.

EXAMPLE 2 Pharmacokinetics of Testosterone and its PhosphocholineDerivative

The phosphocholine derivatives of testosterone (about 5 mg) is dissolvedin 20 ml of buffered saline or in 20 ml of 40% cyclodextran in salineand given orally to human volunteers. Alternatively, testosterone (5 mg)is suspended in a carboxymethyl cellulose suspending media, vortexed andthen given orally. Blood samples will be taken at 30, 60, 120, 240, 360and 720 minutes post-administration and collected in green top tubes.The blood samples are centrifuged and the plasma collected and stored asaliquots in microfuge tubes. The samples are then analyzed fortestosterone in duplicate using a standard RIA kit (Diagnostics ProductsCorp., Tarzana, Calif.).

EXAMPLE 3 Measurement of Bioactivity of Phosphocholine Derivatives

The bioactivity of orally administered estradiol and estradiolphosphocholine will be determined in ovariectomized mice or rats. Inaddition, other animals will be briefly anesthetized and the steroidphosphocholine derivative or the free steroid will be administeredintraperitoneally (IP). After 2 days the animals are sacrificed and the4th and 9th inguinal breast tissue will be isolated. At the same timethe uteri will be isolated and weighed. It is expected that thephosphocholine derivatized steroid will be more active than the parentcompound when administered orally and by IP injection.

Estradiol and its phosphocholine derivative will also be administered bygavage to ovariectomized mice or rats and changes in uterine weight,breast development and estradiol blood levels will be measured.Estradiol will be measured with an RIA kit from Diagnostics ProductsCorp. (Tarzana, Calif.).

Testosterone and its phosphocholine derivative will be administeredorally to castrate male mice or rats and changes in seminal vesicles,prostate size, and levator ani muscle will be determined. Testosteroneblood levels will also be measured by RIA using a kit from DiagnosticsProducts Corp. (Tarzana, Calif.). The compounds will also becharacterized by UKV. Responses will also be measured after IPinjection.

Theophylline and its phosphocholine derivatives will be given orally torats and the blood levels of theophylline will be measured over the next6 hours using an RIA kit (Diagnostics Products Corp., Tarzana, Calif.).

From these tests, the degree to which the phosphocholine derivatives aremore potent than the underivatized parent hormone can be determined;i.e., the same response will be achieved with a smaller dose of thederivatized compound than the 25 parent compound. This will be a measureof greater potency.

EXAMPLE 4 Synthesis of Dehydroepiandrosterione (DHEA)-PhosphocholineDerivative

A dehydroepiandrosterone(DHEA)-phosphocholine derivative was synthesizedas follows: 1 mg of phosphocholine (calcium salt; Sigma Chemical, St.Louis, Mo.) was dissolved in 0.5 ml of formamide (Cat # S-7503; Lot #55HO257; Sigma Chemical) and 0.5 ml of pyridine (Cat # P-4036; Lot #55H1489; Sigma Chemical). 0.025 mCi of (1,2,6,7³H(n)-Dehydroepiandrosterone (Cat # NET814; Lot # 3146097; 89.2 Ci/mmol;Dupont, NEN Products, Boston, Mass.) in 0.025 ml of ethanol was added.The reaction was catalyzed by the addition (as the dry solid) of 5 mg ofdicyclohexylcarbodiimide (Cat # D-3129; Lot # 34hO647; Sigma Chemical).The reaction mixture was incubated overnight at room temperature. In themorning, 9 ml of water was added and the mixture extracted 3 times with10 ml portions of benzene. The benzene extracts were combined andaliquots of both phases were counted in a scintillation counter. Theresults are set forth below:

Aqueous Phase 10,729 cpm (0.01 ml)

Benzene Phase 1,121 cpm (0.01 ml)

The aqueous layer was re-extracted with benzene. The second benzeneextraction yielded 272 cpm (0.01 M1) as a confirmation.

Free DHEA starting material would have been extracted quantitativelyinto benzene with this protocol. The observation that the reactionproduct remains in the aqueous phase confirms its increased hydrophiliccharacteristics.

EXAMPLE 5 DHEA-3-Phosphocholine; Synthesis and Bioactivity

DHEA-phosphocholine (DHEA-PC) was synthesized by sequential reaction ofDHEA, choline, and water with phosphorous oxychloride. The syntheticproduct had the same HPLC retention time and the same mass-spectrum asdid the endogenous, actual compound. It was hydrolyzed by neutralsphingomylenase, but not by acidic sphingomylenase. When human serumextracts were analyzed, mass fragments were detected at the sameretention time as synthetic material. When DHEA-PC was administered tomice, it potentiated dinitrochlorobenzene-induced sensitization asdetailed below.

EXAMPLE 6 DHEA-PC Potentiates DNCB-Induced Immunological Sensitization

The effects of DHEA-PC on cutaneous contact hypersensitivity wasstudied. In this study, mice (Balb/c) were immunologically challengedwith DNCB (2% in ethanol) applied to a 2 cm area on the back. Thesteroid was injected subcutaneously (100 μg/day/mouse) throughout thetwelve-day study period. Ears were rechallenged with DNCB (1% inethanol) on days 7–12 and swelling was measured daily in order toevaluate the effect on the immune system.

During days 7–12, DHEA-PC enhanced the cutaneous hypersensitivity immuneresponse similar to native steroids (DHEA and DHEA-sulfate). Theresponse to these three hormones was not suppressed by dexamethasoneeven though, when administered by itself, dexamethasone suppressed theimmune response below the control. This shows that all three hormonesinduced a similar, high level response.

EXAMPLE 7 Novel Phosphocholine Synthetic Method

DHEA (I) (82.0 g, 0.284 mol, Steraloids, Inc., Wilton, N.H.) wasdissolved in a 5 L, 3 necked, round bottom flask in dry benzene (1.5 L,Fisher, Pittsburgh, Pa.). Gentle heating was applied to facilitate theprocess. Triethylamine (30.3 g, 41.6 mL, 0.30 mol, Aldrich, Milwaukee,Wis.) was added all at once. After the reaction was cooled down to roomtemperature, oxyphosphorus trichloride (43.6 g, 26 mL, 0.284 mol, Fluka,Ronkonkoma, N.Y.) was added in one portion. The mixture was stirredunder nitrogen overnight (12 hours). The precipitate was filtered offvia canula transfer under nitrogen, and washed once with dry benzene(300 mL). To the combined clear benzene solution was added ethyleneglycol (18.6 g, 0.30 mol, Aldrich) and triethylamine (61 g, 0.60 mol,Aldrich). The mixture was stirred rapidly for 16 hours at roomtemperature. Thin layer

chromatography (TLC) (Silica gel, developed with ethyl acetate, Fisher)showed almost complete conversion. The newly formed precipitate wasseparated on a Buchner funnel and washed three times with hot drybenzene (800 mL total). The combined filtrates were evaporated todryness on a rotary evaporator (Buchi, Fisher). The intermediate (II)was a white solid and used for the next step without furtherpurification. An additional amount of the intermediate (II) was obtainedfrom the solid retained by the Buchner funnel by resuspension in waterand vacuum filtration. The combined collected solid was air dried. Theoverall yield of the crude intermediate (II) was virtually quantitative(110 g).

The crude II (2.9 g) was suspended in acetonitrile (25 mL, FisherChemicals). While the mixture was stirred at 50–60° C. with the pressuremaintained through a balloon, trimethylamine (Aldrich) was introduced asthe gas. After the conversion was complete, as indicated by TLCanalysis, the mixture was vacuum filtered, washed repeatedly withacetonitrile and then air dried. The yield was 75% (2.5 g). LC-massspectroscopy (Micromass, Beverly, Mass.) showed a major peak at Rf=9.8min with mass of 454 daltons (M+H), as predicted. DHEA (8.75 g, 0.031mol) was dissolved in benzene and triethylamine (4.45 mL) was added.2-choro-1,3,2-dioxaphospholane-2-oxide (4.54 g, 0.032 mol, Aldrich) wasthen added at room temperature. The reaction mixture was stirred untilcomplete conversion of DHEA to II occurred. The reaction was monitoredby TLC (silica gel, ethyl acetate). After filtration, the solid waswashed with dry benzene. The combined benzene solution was concentratedto give a white solid (II) and

used without further purification.

A sample of II (0.75 g) pared as above was suspended in acetonitrile (10mL) and stirred with heating. Trimethylamine was introduced as a gaswhile the pressure was regulated with a balloon attached to one of thenecks of the flask. When TLC (silica gel, ethyl acetate) showed thedisappearance of II, the addition of gas was stopped. The product (III)was collected by vacuum filtration, washed with additional acetonitrileand air dried. The yield was 0.72 g (83%).

1. A compound having the general formula I:

wherein the LINKER is one or more of the groups selected from the groupconsisting of (i) unsubstituted alkyl, (ii) substituted or unsubstitutedalkenyl, (iii) unsubstituted alkanoyl, (iv) unsubstituted alkenoylwherein the double bond is cis, and (v) (ortho or para)carbonyl-substituted aryl; and wherein the subtituent is each anindependent group or linked together thereby forming a ring; and whereinX is an O or S atom and wherein the substituent is each an independentgroup or linked together thereby forming a ring; and wherein thetherapeutic agent is selected from the group consisting of (i)testosterone, (ii) cardiotonic steroids selected from the groupconsisting of digitoxigenin, digoxigenin and ouabuquenin, (iii)dehydroepiandrosterone (DHEA), (iv) eticholanolone, (v) pregnenolone,(vi) estradiol, (vii) estrone, (viii) dexamethasone, (ix) hydrocortisoneand (x) paclitaxel.
 2. A compound according to claim 1, wherein thetherapeutic agent is Propofol.
 3. A compound according to claim 1,wherein said water-insoluble steroids are selected from the groupconsisting of (i) testosterone, (ii) cardiotonic steroids selected fromthe group consisting of digitoxigenin, digoxigenin and ouabugenin, (iii)dehydroepiandrosterone (DHEA), (iv) etiocholanolone, (v) pregnenolone,(vi) estradiol, (vii) estrone, (viii) dexamethasone and (ix)hydrocortisone.
 4. A compound according to claim 1, further comprisingone or more of the ingredients selected from the group consisting ofpharmaceutically-acceptable carriers, diluents, fillers, salts, buffers,preservatives, antioxidants, a binder, an excipient, a disintegratingagent, a lubricant, and a sweetening agent.
 5. A compound according toclaim 1 incorporated into tablets, capsules or elixirs for oraladministration; suppositories for rectal administration; sterilesolutions or suspensions for injectable administration; or sterilesolutions for ocular or internasal administration.
 6. A compound havingthe general formula I:

wherein the LINKER is a substituted alkanoyl of formulaCR₁R₂—CR₃R₄—CR₅R₆—CO, wherein R₁, R₂, R₃, R₄, R₅, and R₆ are H, andwherein X is O and wherein the therapeutic agent is 2′,6′-diisopropylphenol.
 7. The compound according to claim 1, wherein the therapeuticagent is paclitaxel is propofol.
 8. The composition according to claim2, further comprising a pharmaceutically-acceptable carrier selectedfrom one or more binder, filter, salt, buffer, preservative,antioxidant, disintegrating agent, lubricant or sweetening agent.
 9. Theformulation of claim 8, wherein the physiologically acceptable carriercomprises one or more binder, preservative, stabilizer or flavor.
 10. Acompound having the general formula I:

wherein the LINKER is a substituted alkenoyl of formulaCR₁R₂—CR₃═CR₄—CO, wherein R₁, R₂, R₃, and R₄, are hydrogen, and whereinX is O and wherein the therapeutic agent is 2′,6′-diisopropyl phenol.11. A compound having the general formula I:

wherein the LINKER is of the formula aryl-ortho-CR₃R₄—CR₅R₆—CO, whereinR₃, R₄, R₅, and R₆, are hydrogen, and wherein X is O and wherein thetherapeutic agent is 2′,6′-diisopropyl phenol.
 12. A compound having thegeneral formula 1:

wherein the LINKER is a substituted alkenyl of formula CR₁R₂—CR₃═CR₄—CO,wherein R₁, R₃, and R₄, are hydrogen and wherein the double bond istrans, and wherein X is O and wherein the therapeutic agent is2′,6′-diisopropyl phenol.